Method and composition for determining specific antibody responses to species of filovirus

ABSTRACT

The disclosure relates to compositions, assays, methods and kits comprising one or more amino acid sequences of a filovirus protein, or a fragment thereof, which find use in the detection of a filovirus infection and/or the presence of antibodies specific for a filovirus in a biological sample.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a National Stage Application under 35 U.S.C. § 371 of International Application No. PCT/US2015/34080, filed Jun. 3, 2015, which claims priority to U.S. Provisional Patent Application No. 62/007,195, filed on Jun. 3, 2014, which is incorporated by reference herein in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

The invention was made with support from the National Institute of Allergy and Infectious Diseases (R01AI96215), and the Defense Threat Reduction Agency (contract CB3948).

SEQUENCE LISTING

This application is submitted with a Sequence Listing text file that serves as both the computer readable form (CRF) and the paper copy required under 37 C.F.R. § 1.821. The sequence listing, which is incorporated by reference, includes a the entitled “SequenceListing.txt” which is 92 kilobytes in size and was created on Jun. 1, 2015.

BACKGROUND

Filoviruses, which include marburgviruses and ebolaviruses, cause severe viral hemorrhagic fever. The first outbreak of Marburg virus was recorded in 1967 in Germany and Yugoslavia, and was traced to infected African green monkeys from Uganda (1). Since then, major outbreaks of marburgviruses have occurred in sub-Saharan Africa. The first outbreaks of Ebola virus were documented in Sudan and The Democratic Republic of Congo in 1976 (2, 3). Because no licensed therapeutics or vaccines are currently available, cycles of filovirus outbreaks are a major concern in biodefense as well as public health. Filoviral hemorrhagic fever is characterized by rapid disease onset and mortality rates of up to 90% (4). Following an incubation period that can range from 2-21 days, infected patients commonly develop non-specific flulike symptoms of fever, vomiting, loss of appetite, headache, abdominal pain, fatigue, and diarrhea, while bleeding occurs in a smaller number of infections (1, 3, 5). Case fatalities are associated with reduced adaptive immune responses (6, 7) and the release of high levels of immune response mediators (8-10) that contribute to vascular dysfunction, coagulation disorders, shock and eventual multi-organ failure (2).

There is a persistent need for sensitive and reliable serological approaches for examining filoviral infections. Because genetic material from the pathogen is often missing, antibody detection methods are indispensable, especially for examining nonviremic patients and for disease surveillance. While ELISAs for detecting specific IgG and IgM based on live virus preparation were previously developed (11-13), the need for BSL-4 labs and associated safety issues are major limitations. Serological assays based on recombinant filovirus antigens are alternatives that do not require infectious agents, and several ELISAs were reported (14-18). For example, Nakayama and coworkers developed a GP-based ELISA representative of all six species of filoviruses and analyzed human patient sera from Ebola and Marburg virus outbreaks (Nakayama et al, 2010). However, these previous methods have only addressed a limited number of antigens and species of filoviruses. The Filoviridae family includes one species of Marburg virus (Marburg marburgvirus), with five species of Ebola virus (Sudan, Zaire, Reston, Bundibugyo, and Taï Forest ebolavirus) that are each a cause of severe hemorrhagic fevers in primates including humans (2). Further complicating assay development, the single-stranded, negative-sensed RNA genome (˜19 kB) encodes seven structural proteins (1, 19, 20) that are each potential antigens: the nucleoprotein (NP), virion protein 35 (VP35), VP40, glycoprotein (GP), VP30, VP24, and RNA-dependent RNA polymerase (L). Major functions of each component of the viral proteome were previously characterized. The RNA genome is encapsulated by NP, and the ribonucleoprotein complex is associated with VP35, VP30, and L (21, 22). Transcription and replication of the viral genome requires L, NP, and VP35 (23), while transcription for Ebola virus, but not Marburg virus, requires VP30 as an additional co-factor (24, 25). VP40 is a matrix protein critical for virion assembly as well as budding from infected cells (26, 27), and VP24 appears to play a role in nucleocapsid assembly and inhibition of interferon signaling (28-30). Unlike Marburg GP, Ebola GP is expressed following RNA editing, while the unedited transcript encodes a soluble GP that is released from infected cells (31, 32). Further, trimeric GP complexes on the virion surface are receptors for fusion and entry into the host cell (33-35).

Compositions and methods that can provide for a fast, accurate, and comprehensive serological analysis would help faciliate identification and diagnosis of filovirus infection (e.g., one or more filovirus antibodies in a sample) in the general human population as well as potentially animal populations. Such compositions and methods would provide an important tool in the detection, management, and containment of filovirus outbreaks, and ultimately help to reduce mortality rates and public panic that are associated with these hemorrhagic fever viruses.

SUMMARY

In one aspect the disclosure provides a detection agent comprising one or more amino acid sequences of a filovirus protein, or a fragment thereof, and a substrate, wherein the one or more amino acid sequences of the filovirus protein is attached to the substrate.

In embodiments, the one or more amino acid sequences of a filovirus protein is from a filovirus selected from Marburg marburgvirus, Sudan ebolavirus, Zaire ebolavirus, Reston ebolavirus, Bundibugyo ebolavirus, and Taï Forest ebolavirus. In some embodiments the one or more amino acid sequences of a filovirus protein, or fragment thereof, is selected from nucleoprotein (NP), virion protein 40 (VP40), glycoprotein (GP), virion protein (VP35), virion protein (VP30), virion protein (VP24), RNA-dependent RNA polymerase (L), or any combination thereof of the same or different filovirus. In some embodiments the detection agent comprises from two or more amino acid sequences to twenty or more amino acid sequences (e.g., 2, 3, 4, 5, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, or more amino acid sequences) from the same or different filovirus. In some embodiments the detection agent comprises at least three different amino acid sequences of at least three different filovirus proteins, or fragments thereof, and in further embodiments, the at least three different proteins may comprise NP, VP40, and GP, or fragments thereof. In some embodiments the detection agent comprises a protein having at least 90% sequence identity to the sequence of a filovirus protein.

In some embodiments, the detection agent may comprise a substrate is selected from the group consisting of a microarray, microparticles, and nanoparticles, and such substrates may be made of materials including glasses, plastics, chemical/biological polymers, metal (magnetic and non-magnetic) semiconductors, ceramics, and the like. In some embodiments the detection agent is a microarray.

In certain embodiments, the one or more amino acid sequences of a filovirus protein may be provided as a recombinant protein or a fragment thereof.

In an aspect the disclosure provides a method for detecting the presence of filovirus-specific antibody in biological sample obtained from a subject comprising:

-   -   (a) incubating the biological sample with a detection agent         comprising one or more amino acid sequences of a filovirus         protein, or a fragment thereof, attached to a substrate under         conditions that allow binding of the filovirus-specific antibody         to the detection agent; and     -   (b) detecting the filovirus-specific antibody bound to detection         agent.

In another aspect the disclosure provides a method for identifying a subject infected with a filovirus, comprising:

determining whether a filovirus-specific antibody is present in a sample obtained from the subject, wherein the determining comprises:

-   -   (a) incubating the biological sample with a detection agent         comprising one or more amino acid sequences of a filovirus         protein, or a fragment thereof, attached to a substrate under         conditions that allow binding of the filovirus-specific antibody         to the detection agent; and     -   (b) detecting the filovirus-specific antibody bound to the         detection agent,

wherein the detection of the filovirus-specific antibody identifies that the subject is infected with a filovirus.

In a further aspect, the disclosure provides a method for identifying whether a subject is infected with a filovirus, comprising:

determining whether an antibody to the filovirus is present in a sample obtained from the subject, wherein the determining comprises:

-   -   (a) contacting the sample with at least one protein, or a         fragment thereof, from the filovirus to which the antibody can         specifically bind; and     -   (b) detecting specific binding between the at least one protein         and the antibody,

wherein the detection of specific binding identifies that the subject is infected with a filovirus.

In various embodiments of the above aspects relating to methods, the filovirus may be selected from the group consisting of Marburg marburgvirus, Sudan ebolavirus, Zaire ebolavirus, Reston ebolavirus, Bundibugyo ebolavirus, and Taï Forest ebolavirus or any combination thereof. In embodiments the methods comprise one or more amino acid sequences of a filovirus protein, or a fragment thereof, comprises nucleoprotein (NP), virion protein 40 (VP40), glycoprotein (GP), virion protein (VP35), virion protein (VP30), virion protein (VP24), RNA-dependent RNA polymerase (L), or any combination thereof.

In some embodiments the detection agent comprises from two or more amino acid sequences to twenty or more amino acid sequences (e.g., 2, 3, 4, 5, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acid sequences) from the same or different filovirus or filovirus protein. In some embodiments the detection agent comprises at least three different amino acid sequences of at least three different filovirus proteins, or fragments thereof, and in further embodiments, the at least three different proteins may comprise NP, VP40, and GP, or fragments thereof. In some embodiments the detection agent comprises a protein having at least 90% sequence identity to the sequence of a filovirus protein.

In further embodiments, the methods can comprise the comparison the amount of an filovirus-specific antibody detected according to the method with one or more control values (e.g., measuring the amount of signal generated from incubating the detection agent with a normal (healthy and/or uninfected) biological sample).

In other aspects the disclosure provides a kit comprising, the detection agent as described herein; at least one reagent that can detect a filovirus-specific antibody bound to the detection agent; and instructions for use of the kit.

In yet a further aspect, the disclosure provides a method for making the detection agent described herein, the method comprising:

-   -   expressing one or more recombinant polynucleotide sequences         encoding an amino acid sequence of a filovirus protein, or a         fragment thereof in an expression system; and     -   fixing the encoded amino sequence of a filovirus protein, or a         fragment thereof, on a surface of the substrate.

In embodiments of this aspect, the method may comprise an recombinant expression system including a prokaryotic cell, a eukaryotic cell, or in vitro translation, or any combination thereof. In further embodiments, the prokaryotic cell may comprise a bacterium such as, for example, E. coli. In other embodiments, the eukaryotic cell may be selected from the group consisting of yeast, an insect cell, and a mammalian cell.

Other aspects and embodiments will be apparent to those of skill in the art in view of the description and illustrative Examples that follow.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1A-1C. Validation of filovirus microarray using control antibodies. A panel of antibodies against A) Marburg virus, B) Zaire ebolavirus, and C) Sudan ebolavirus proteins were tested on printed microarrays. All antibodies are mouse monoclonal except for anti-Zaire-NP and -VP40 which are both rabbit polyclonal. Bound antibodies were detected fluorescently on a microarray scanner. Background-corrected fluorescence intensities were averaged across technical replicates on the microarrays. Bars represent mean fluorescence (RFU)±SEM. All GP ΔTMs were expressed in insect cells except for Marburg-GP ΔTM (Musoke) which was expressed in mammalian cells. Bundibugyo (B), Taï Forest (T), Marburg (M), Reston (R), Sudan (S), and Zaire (Z).

FIG. 2A-2C. IgG antibody response detected using filovirus microarray. Naïve, post-immunization (immunized), and post-viral challenge (challenged) sera from A) Zaire ebolavirus and B) Marburg virus animal studies were applied to assembled microarrays. Bound IgG antibodies were detected fluorescently on a microarray scanner. Following data pre-processing, normalized fluorescence signals were averaged across the five animals in each study. Bars represent normalized mean fluorescence (RFU)±SEM. The cutoff line represents two standard deviations above the mean antibody signal observed in naïve sera. For each antigen-antibody response, paired t-test was done for naïve versus immunized and naïve versus challenged sera. Unless indicated with an ‘*’, all immunized and challenged samples above the cutoff line were found to have significant antibody increases (p<0.05) in comparison with the naïve samples. Bundibugyo (B), Taï Forest (T), Marburg (M), Reston (R), Sudan (S), and Zaire (Z). C) Side-by-side comparison of GP-specific IgG signals in challenged sera from Zaire ebolavirus and Marburg marburgvirus studies.

FIG. 3A-B. Comparison of antibody signals between E. coli- and eukaryotic cell-expressed GP. Data was acquired and analyzed in a similar manner as in FIG. 2. Bars represent normalized mean fluorescence (RFU)±SEM. All GP-mucins were expressed in E. coli. All GP ΔTM were expressed in insect cells except for Marburg GP ΔTM (Musoke) which was expressed in mammalian cells. A) Zaire ebolavirus study. B) Marburg marburgvirus study.

FIG. 4A-B. Microarray schematic and representative scanned fluorescent image of processed microarray. A) Slide schematic shows multiple 12×12 microarrays printed on a slide, along with an enlarged layout of an individual microarray. Table below provides the sample identity for each triplicate microarray spots. Red circles represent fluorescently-labeled streptavidin serving as reference markers for orientation purposes. Bundibugyo (B), Taï Forest (T), Marburg (M), Reston (R), Sudan (S), and Zaire (Z). B) Representative GenePix®-scanned image of microarray processed with naïve (left) and post-challenge sera (right) from the ZEBOV vaccine study.

FIG. 5. Scatter plot of individual antibody responses for primates challenged with Zaire ebolavirus. Bound IgG were detected fluorescently on a microarray scanner. Background-corrected fluorescence intensities were averaged across technical replicates on the microarrays. Pre-vaccination, dark circles; post-vaccination, open circles; post-viral challenge, dark triangles. Bundibugyo (B), Taï Forest (T), Marburg (M), Reston (R), Sudan (S), and Zaire (Z).

FIG. 6. Scatter plot of individual antibody responses for primates challenged with Marburg marburgvirus. Bound IgG were detected fluorescently on a microarray scanner. Background-corrected fluorescence intensities were averaged across technical replicates on the microarrays. Pre-vaccination, dark circles; post-vaccination, open circles; post-viral challenge, dark triangles. Bundibugyo (B), Taï Forest (T), Marburg (M), Reston (R), Sudan (S), and Zaire (Z).

FIG. 7. IgM signals for sera collected from a single animal in the ZEBOV challenge groups. Bound antibodies were detected fluorescently on a microarray scanner. Background-corrected fluorescence intensities were averaged across technical replicates on the microarrays. Bundibugyo (B), Taï Forest (T), Marburg (M), Reston (R), Sudan (S), and Zaire (Z).

FIG. 8A-C. Phylogenic relationships between filovirus strains based on amino acid sequences of NP and GP mucin-like domain. Separate dendrograms representative of sequence similarity between filovirus strains were derived based on amino acid sequences of A) a conserved region of 406 residues of NP (BDBV, TAFV, RESTV, SUDV, EBOV-residues 20-425; MARV-residues 2-407), and B) the highly unconserved mucin-like domain of GP, consisting of 33 residues at the N-terminus of the domain region (BDBV, TAFV, RESTV-residues 2-34; EBOV and MARV-residues 1-33). Maximum likelihood tress are shown with bootstrap values (out of 1000 replicates) shown at internal nodes. (C) Sequence identity matrix comparison of filovirus NP and GP mucin-like domain amino acid sequences. Full length sequences of NP and the GP mucin-like domain were used to generate percent identity matrices. NP (light grey, upper triangle) is highly conserved among ebolavirus strains and more divergent from MARV, while the GP mucin-like domain is highly variable and exhibits minimal sequence identify among all filovirus strains examined.

FIG. 9. Antibody reactivity to filoviral proteins in a cohort of ebola and marburg survivors. Heat map displaying IgG reactivity associated with filoviral infection and controls. Hierarchical clustering by Euclidean distance average linkage method was used to visualize protein microarray results. Normalized and log 2-transformed data was applied for creating the heat map. The IDs of proteins are listed in the rows (*insect and**mammalian expressed), the cells represent individual sera samples, and the survivor and control groups are listed on the bottom of the colored bars. The blue bars show ACAM2000 healthy controls, the purple bars healthy controls from Uganda and the green bars three MARV, SUDV and BDBV survivor groups.

FIG. 10. Ebola and Marburg survivors convalescent IgG responses to autologous GP-mucin, NP and VP40 recombinant proteins. Panel a shows reactivity to BDBV, panel b to SUDV-Gulu and panel c to MARV antigens by survivors. The filled circles denote survivors and open circles controls. Each circle corresponds to individual sera sample and the red line represents geometric mean of all samples in each group. Statistical analysis was performed using Prospector software for comparing survivor Vs. controls. Significant differences between the two groups in terms of p values are shown as *p,0.05, **p,0.01, ***p,0.001

FIG. 11. Survivor sera antibody cross-reactivity to filoviral heterologous compared to autologous antigens. Each bar represents the mean antibody binding of all samples in a survivor group (MARV, SUDV or BDBV) expressed as relative fluorescence units (RFU).

FIG. 12. Antibody responses by a replication defective recombinant EBOV/SUDV GP vaccinated subject to filoviral proteins. The bars represent mean values of four replicates (* insect and ** mammalian expressed).

FIG. 13. Comparison antibody responses to GP proteins.

FIG. 14. List of significant antibody responses to autologous and heterologous antigens and their p-values. The three survivor groups are on the left column and the antigen hits are listed on the right cells.

DETAILED DESCRIPTION

As discussed in further detail below, the inventors have developed compositions of matter (e.g., detection agents, kits, etc.) and methods that can provide for fast, accurate, comprehensive and convenient (e.g., point-of-care assays) detection of filovirus antibodies in biological samples such as, for example, sera. The efficacy of the compositions and methods relating to the general technology is demonstrated through the illustrative embodiments disclosed in the Examples. For example, certain embodiments provide for the preparation and use of protein microarrays as the detection agent disclosed and described herein, and by including one or more amino acid sequences of at least one filovirus protein (e.g., GP or fragments thereof), or combinations of filovirus proteins and/or fragments thereof (e.g., NP, GP, and VP40, as discussed below), allow for the detection of an antibody to one or more filovirus (e.g., Ebola and/or Marburg virus) species. Further, and unexpectedly, the inventors have identified that in particular embodiments one or more amino acid sequences of filovirus proteins and/or fragments thereof, may be recombinantly expressed in expression systems, including prokaryotic cells, (e.g., E. coli) without any loss of filovirus-specific antibody binding activity or specificity.

In practicing the technology disclosed herein, many conventional techniques in molecular biology, protein biochemistry, cell biology, immunology, and microbiology are used. These techniques are well-known and are explained in, e.g., Current Protocols in Molecular Biology, Vols. I-III, Ausubel, Ed. (1997); Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989); DNA Cloning: A Practical Approach, Vols. I and H, Glover, Ed. (1985); Transcription and Translation, Hames & Higgins, Eds. (1984); Animal Cell Culture, Freshney, Ed. (1986); Immobilized Cells and Enzymes (IRL Press, 1986); Perbal, A Practical Guide to Molecular Cloning; the series, Meth. Enzymol., (Academic Press, Inc., 1984); Gene Transfer Vectors for Mammalian Cells, Miller & Calos, Eds. (Cold Spring Harbor Laboratory, NY, 1987); and Meth. Enzymol., Vols. 154 and 155, Wu & Grossman, and Wu, Eds., respectively. Methods to detect and measure the levels of protein complexes are well-known in the art and include ELISA assays, and co-immunoprecipitation assays.

All references referred to within the body of the application are incorporated herein by reference in their entirety.

Definitions

Virus as used herein refers to a small infectious agent that replicates only inside the living cells of other organisms. Viruses can infect all types of life forms, from animals and plants to microorganisms, including bacteria and archaea. Virus particles (known as virions) typically consist of two or three parts: i) the genetic material made from either DNA or RNA, long molecules that carry genetic information; ii) a protein coat that protects these genes; and in some cases iii) an envelope of lipids that surrounds the protein coat when they are outside a cell. The shapes of viruses range from simple helical and icosahedral forms to more complex structures. The average virus is about one one-hundredth the size of the average bacterium.

Filoviruses generally refer to viruses of the viral family called Filoviridae and infection can cause severe hemorrhagic fever in humans and nonhuman primates. So far, three members of this virus family have been identified: Marburgvirus, Ebolavirus, and Cuevavirus. Five species of Ebolavirus have been identified: Taï Forest (formerly Ivory Coast), Sudan, Zaire, Reston and Bundibugyo. Ebola-Reston is the only known Filovirus that does not cause severe disease in humans; however, it can still be fatal in monkeys and it has been recently recovered from infected swine in South-east Asia. Structurally, filovirus virions (complete viral particles) may appear in several shapes, a biological feature called pleomorphism. These shapes include long, sometimes branched filaments, as well as shorter filaments shaped like a “6”, a “U”, or a circle. Viral filaments may measure up to 14,000 nanometers in length, have a uniform diameter of 80 nanometers, and are enveloped in a lipid (fatty) membrane. Each virion contains one molecule of single-stranded, negative-sense RNA. New viral particles are created by budding from the surface of their hosts' cells; however, filovirus replication strategies are not completely understood.

The genus Ebolavirus (or “Ebola” or “Ebola virus”) is a virological taxon included in the family Filoviridae, order Mononegavirales. The members of this genus are generally referred to as ebolaviruses. The five known virus species are named for the region where each was originally identified: Bundibugyo ebolavirus, Reston ebolavirus, Sudan ebolavirus, Taï Forest ebolavirus (originally Côte d'Ivoire ebolavirus), and Zaire ebolavirus.

The genus Marburgvirus (or “Marburg” or “Marburg virus”) refers to the species, Marburg marburgvirus, which includes two known marburgviruses, Marburg virus (MARV) and Ravn virus (RAVV). Both viruses cause Marburg virus disease, a form of hemorrhagic fever, in humans and nonhuman primates.

Cuevavirus is a genus in the family Filoviridae that has one identified species, Lloviu cuevavirus (LLOV or “cueva virus”). Studies indicate that LLOV is a distant relative of the more widely known Ebola and Marburg viruses.

DNA is used herein (Deoxyribonucleic acid) is a molecule that encodes the genetic instructions used in the development and functioning of all known living organisms and many viruses. DNA is a nucleic acid; alongside proteins and carbohydrates, nucleic acids compose the three major macromolecules essential for all known forms of life. Most DNA molecules consist of two biopolymer strands coiled around each other to form a double helix. The two DNA strands are known as polynucleotides since they are composed of simpler units called nucleotides. Each nucleotide is composed of a nitrogen-containing nucleobase—either guanine (G), adenine (A), thymine (T), or cytosine (C)—as well as a monosaccharide sugar called deoxyribose and a phosphate group. The nucleotides are joined to one another in a chain by covalent bonds between the sugar of one nucleotide and the phosphate of the next, resulting in an alternating sugar-phosphate backbone. According to base pairing rules (A with T and C with G), hydrogen bonds bind the nitrogenous bases of the two separate polynucleotide strands to make double-stranded DNA.

Proteins are large biological molecules, or macromolecules, consisting of one or more long chains of amino acid residues (e.g., “peptide”, “polypeptide”, or “amino acid sequence”). Proteins perform a vast array of functions within living organisms, including catalyzing metabolic reactions, replicating DNA, responding to stimuli, and transporting molecules from one location to another. Structural differences in proteins typically arise based on differences between secondary structure (primarily sequence of amino acids), which is dictated by the nucleotide sequence of their genes, and tertiary and/or quaternary structure (protein folding, three-dimensional structure, and domain interactions), all of which can determine activity.

An antigen (Ag), abbreviation of antibody generator, is any structural substance which serves as a target for the receptors of an adaptive immune response, TCR or BCR or its secreted form antibody, respectively. Each antibody is specifically selected after binding to a certain antigen because of random somatic diversification in the antibody complementarity determining regions (a common analogy used to describe this is the fit between a lock and a key). In summary, an antigen is a molecule that binds to Ag-specific receptors but cannot induce an immune response in the body by itself. Antigen was originally a structural molecule that binds specifically to the antibody, but the term now also refers to any molecule or a linear fragment that can be recognized by highly variable antigen receptors (B-cell receptor or T-cell receptor) of the adaptive immune system.

An antibody (AB), also known as an immunoglobulin (Ig), is a large, Y-shape protein produced by plasma cells that is used by the immune system to identify and neutralize pathogens such as bacteria and viruses. The antibody recognizes a unique molecule of the harmful agent, called an antigen, via the variable region. Each tip of the “Y” of an antibody contains a paratope that is specific for one particular epitope (similarly analogous to a key) on an antigen, allowing these two structures to bind together with precision. Using this binding mechanism, an antibody can tag a microbe or an infected cell for attack by other parts of the immune system, or can neutralize its target directly (for example, by blocking a part of a microbe that is essential for its invasion and survival). The ability of an antibody to communicate with the other components of the immune system is mediated via its Fc region (located at the base of the “Y”), which contains a conserved glycosylation site involved in these interactions. The production of antibodies is the main function of the humoral immune system.

A microarray as used herein refers to the technology generally identified as a multiplex lab-on-a-chip. Typically a microarray comprises a 2D array on a solid substrate (usually a glass slide or silicon thin-film cell) that assays large amounts of biological material using high-throughput screening miniaturized, multiplexed and parallel processing and detection methods. A protein microarray (or protein chip) is a high-throughput method used to track the interactions and activities of proteins, and to determine their function, and determining function on a large scale. Its main advantage lies in the fact that large numbers of proteins can be tracked in parallel. The chip consists of a support surface such as a glass slide, nitrocellulose membrane, bead, or microtitre plate, to which an array of capture proteins is bound. Probe molecules, typically labeled with a fluorescent dye, may added to the array. Any reaction between the probe and the immobilized protein emits a fluorescent signal that is read by a laser scanner. Protein microarrays may be rapid, automated, economical, and highly sensitive, consuming small quantities of samples and reagents. Methodology relating to protein microarrays was introduced as early as 1983, illustrated using antibody-based microarrays (also referred to as antibody matrix).

Mucin are a family of high molecular weight, heavily glycosylated proteins (glycoconjugates) produced by epithelial tissues in most organisms of Kingdom Animalia. Mucins' key characteristic is their ability to form gels; therefore they are a key component in most gel-like secretions, serving functions from lubrication to cell signalling to forming chemical barriers. They often take an inhibitory role. Some mucins are associated with controlling mineralization, including nacre formation in mollusks, calcification in echinoderms and bone formation in vertebrates. They bind to pathogens as part of the immune system. Overexpression of the mucin proteins, especially MUC1, is associated with many types of cancer. Although some mucins are membrane-bound due to the presence of a hydrophobic membrane-spanning domain that favors retention in the plasma membrane, most mucins are secreted onto mucosal surfaces or secreted to become a component of saliva.

The cell is the basic structural, functional, and biological unit of all known living organisms. Cells are the smallest unit of life that can replicate independently, and are often called the “building blocks of life”. A prokaryote is a single-celled organism that lacks a membrane-bound nucleus (karyon), mitochondria, or any other membrane-bound organelles. A eukaryote is any organism whose cells contain a nucleus and other organelles enclosed within membranes.

Recombinant protein is a protein produced by a recombinant DNA that encodes for the protein sequence. Recombinant DNA (rDNA) molecules are DNA molecules formed by laboratory methods of genetic recombination (such as molecular cloning) to bring together genetic material from multiple sources, creating sequences that would not otherwise be found in biological organisms. Once a recombinant DNA is inserted into bacteria, these bacteria will make protein based on this recombinant DNA. This protein is known as “Recombinant protein”.

Infection is the invasion of an organism's body tissues by disease-causing agents, their multiplication, and the reaction of host tissues to these organisms and the toxins they produce. Infectious disease, also known as transmissible disease or communicable disease is illness resulting from an infection. Infections discussed herein are typically caused filoviruses.

Detection Agent

In a general aspect, the disclosure provides a detection agent comprising one or more amino acid sequences of a filovirus protein, or a fragment thereof, and a substrate wherein the one or more amino acid sequences of the filovirus protein is attached to substrate. In certain embodiments, the one more amino acid sequences of a filovirus protein may comprise a sequence of a protein from a filovirus selected from Marburg marburgvirus, Sudan ebolavirus, Zaire ebolavirus, Reston ebolavirus, Bundibugyo ebolavirus, and Taï Forest ebolavirus.

In some embodiments, the one or more amino acid sequences of a filovirus protein, or fragment thereof, may comprise a sequence from a nucleoprotein (NP), virion protein 40 (VP40), glycoprotein (GP), virion protein (VP35), virion protein (VP30), virion protein (VP24), RNA-dependent RNA polymerase (L), or a fragment thereof, or any combination thereof. Any amino acid sequence that provides for binding and recognition of a filovirus specific antibody may be used in connection with the detection agent. In some embodiments, the amino acid sequence may exhibit little to no cross-reactivity to filovirus specific antibodies that are directed to a particular type of filovirus or a particular filovirus protein. In certain embodiments the one or more amino acid sequences of a filovirus protein comprises GP, or fragment thereof. The GP or fragment thereof may comprise a mucin-like domain fragment of GP (GP-mucin) or a GP ectodomain (GPΔTM). While the detection agent comprises at least one amino acid sequence of a filovirus protein, it may also comprise a plurality of such amino acid sequences. In some embodiments the detection agent comprises from two or more amino acid sequences to twenty or more amino acid sequences (e.g., 2, 3, 4, 5, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acid sequences) that may be selected from the same or different filovirus and/or the same or different filovirus protein. As a further example, in some embodiments, such as those illustrated in the non-limiting Examples, the detection agent may include at least three different amino acid sequences of at least three different filovirus proteins, or fragments thereof (e.g., NP, VP40, and GP, or fragments thereof).

In some embodiments, the amino acid sequences comprising the detection agent can comprise a sequence that is not identical to the protein sequence from which it is derived. Some minor changes in the primary amino acid sequence and/or post-translational modification and processing of the sequence may be allowable as long as the sequence modification does not interfere with the ability of the filovirus-specific antibody to bind. In some embodiments, the detection agent can comprise one or more amino acid sequences having at least 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) to the filovirus protein from which it is derived. In some embodiments, the amino acid sequences may comprise an NP sequence having at least 90% sequence identity to the sequence selected from the group consisting of SEQ ID NO:4 (Zaire NP); SEQ ID NO: 10 (Sudan NP); SEQ ID NO: 16 (Bundibugyo NP); SEQ ID NO: 22 (Taï Forest NP); SEQ ID NO: 28 (Reston NP); and SEQ ID NO: 34 (Marburg NP); a VP40 sequence having at least 90% sequence identity to the sequence selected from the group consisting of SEQ ID NO: 2 (Zaire VP40); SEQ ID NO: 8 (Sudan VP40); SEQ ID NO: 14 (Bundibugyo VP40); SEQ ID NO: 20 (Taï Forest VP40); SEQ ID NO: 26 (Reston VP40); and SEQ ID NO: 32 (Marburg VP40); and/or a GP-mucin domain having at least 90% sequence identity to the sequence selected from the group consisting SEQ ID NO: 6 (Zaire GP-mucin); SEQ ID NO: 12 (Sudan GP-mucin); SEQ ID NO: 18 (Bundibugyo GP-mucin); SEQ ID NO: 24 (Taï Forest GP-mucin); SEQ ID NO: 30 (Reston GP-mucin); and SEQ ID NO: 36 (Marburg GP-mucin). In further embodiments, the detection agent may comprise an NP sequence selected from the group consisting of SEQ ID NO: 4 (Zaire NP); SEQ ID NO: 10 (Sudan NP); SEQ ID NO: 16 (Bundibugyo NP); SEQ ID NO: 22 (Taö Forest NP); SEQ ID NO: 28 (Reston NP); and SEQ ID NO: 34(Marburg NP); a VP40 selected from the list consisting of SEQ ID NO: 2 (Zaire VP40); SEQ ID NO: 8 (Sudan VP40); SEQ ID NO: 14 (Bundibugyo VP40); SEQ ID 20 (Taï Forest VP40); SEQ ID NO: 26 (Reston VP40); SEQ ID NO: 32 (Marburg VP40); and/or a GP-mucin domain selected from the group consisting of SEQ ID NO: 6 (Zaire GP-mucin); SEQ ID NO: 12 (Sudan GP-mucin); SEQ ID NO: 18 (Bundibugyo GP-mucin); SEQ ID NO: 24 (Taï Forest GP-mucin); SEQ ID NO: 30 (Reston GP-mucin); and SEQ ID NO: 36 (Marburg GP-mucin).

As discussed herein, in certain embodiments the substrate may comprise a bead or particle (e.g., microparticle or nanoparticle). In other embodiments the substrate may comprise a substantially planar surface with a plurality of addressable locations that are each associated with a known amino acid sequence of a filovirus protein, and optionally one or more control locations (e.g., a microarray). As discussed further, in some embodiments, the one or more amino acid sequences of a filovirus protein is provided as a recombinant protein or a fragment thereof.

In certain embodiments, the detection agent can comprise any one or more controls such as, for example, a positive control, a negative control, an assay standard, an assay calibrator, a competition assay ligand, a labeled peptide or a solid-phase capture agent. Similarly in some embodiments the amino acid sequences (including any controls as well as the amino acid sequence(s) of a filovirus protein(s)) may comprise a synthetic peptide, a recombinant polypeptide, a substantially purified natural polypeptide, a peptide mimetic, an oligonucleotide aptamer, a polypeptide aptamer, any fragment thereof that can be bound by a filovirus-specific antibody and any combinations thereof. In certain embodiments the detection agent comprises an amino acid sequence of a filovirus protein that is recombinantly produced.

In certain embodiments the detection agent comprises a protein microchip or microarray comprising one or more amino acid sequences of a filovirus protein, or a fragment thereof, and a substrate to which the one or more amino acid sequences are attached. In these embodiments, the microarrays may be useful in a variety of applications including large-scale and/or high-throughput screening for a filovirus-specific antibody that bind to the microarray. In other embodiments, the microarray can be used to identify compound or agents that are capable of modulating the interactions between the filovirus-specific antibody and the amino acid sequence to which it binds.

Regardless of the particular format (e.g., microarray-based or particle-based), the detection agent may be prepared according to any of the techniques described herein or otherwise known in the art. For example, in embodiments relating to a protein microarray, the array can be prepared in a number of methods known in the art. For example, glass microscope slides are treated with an aldehyde-containing silane reagent. Small volumes of protein samples in a phosphate-buffered saline with 40% glycerol may be spotted onto the treated slides using a high-precision contact-printing robot. After incubation, the slides are immersed in a buffer containing for example, bovine serum albumin (BSA) to quench the unreacted aldehydes and to form a BSA layer that functions to prevent non-specific protein binding in subsequent applications of the array/microchip. Alternatively, proteins or protein complexes can be attached to a BSA-NHS slide by covalent linkages. BSA-NHS slides are fabricated by first attaching a molecular layer of BSA to the surface of glass slides and then activating the BSA with N,N′-disuccinimidyl carbonate. As a result, the amino groups of the lysine, aspartate, and glutamate residues on the BSA are activated and can form covalent urea or amide linkages with protein samples spotted on the slides. Alternatively, arrays may be prepared as discussed in the illustrative Examples below.

Methods

In further aspects, the disclosure relates to a number of methods. In an aspect the disclosure provides a method for detecting the presence of filovirus-specific antibody in biological sample obtained from a subject including: (a) incubating the biological sample with a detection agent comprising one or more amino acid sequences of a filovirus protein, or a fragment thereof, attached to a substrate under conditions that allow binding of the filovirus-specific antibody to the detection agent; and (b) detecting the filovirus-specific antibody bound to detection agent.

In another aspect, the disclosure provides a method for identifying a subject infected with a filovirus, comprising determining whether a filovirus-specific antibody is present in a sample obtained from the subject, wherein the determining includes: (a) incubating the biological sample with a detection agent comprising one or more amino acid sequences of a filovirus protein, or a fragment thereof, attached to a substrate under conditions that allow binding of the filovirus-specific antibody to the detection agent; and (b) detecting the filovirus-specific antibody bound to the detection agent, wherein the detection of the filovirus-specific antibody identifies that the subject is infected with a filovirus.

In yet another aspect, the disclosure provides a method for identifying whether a subject is infected with a filovirus, comprising: determining whether an antibody to the filovirus is present in a sample obtained from the subject, wherein the determining includes: (a) contacting the sample with at least one protein, or a fragment thereof, from the filovirus to which the antibody can specifically bind; and (b) detecting specific binding between the at least one protein or the fragment thereof and the antibody, wherein the detection of specific binding identifies that the subject is infected with a filovirus.

Similarly to the embodiments relating to the detection agent discussed herein, in some embodiments of the aspects relating to the above methods, the filovirus may be selected from the group consisting of Marburg marburgvirus, Sudan ebolavirus, Zaire ebolavirus, Reston ebolavirus, Bundibugyo ebolavirus, and Taï Forest ebolavirus.

In some embodiments the at least one protein or fragment thereof comprises nucleoprotein (NP), virion protein 40 (VP40), glycoprotein (GP), virion protein (VP35), virion protein (VP30), virion protein (VP24), RNA-dependent RNA polymerase (L), or any combination thereof, from any one or more filoviruses. Any amino acid sequence that provides for binding and recognition of a filovirus specific antibody may be used in connection with the method. In some embodiments, the amino acid sequence may exhibit little to no cross-reactivity to filovirus specific antibodies that are directed to a particular type of filovirus or a particular filovirus protein. In certain embodiments the one or more amino acid sequences of a filovirus protein comprises GP, or fragment thereof. The GP or fragment thereof may comprise a mucin-like domain fragment of GP (GP-mucin) or a GP ectodomain (GPΔTM). While the methods comprise at least one amino acid sequence of a filovirus protein, the methods may also comprise a plurality of such amino acid sequences from a single filovirus protein, and/or a plurality of filovirus proteins, and/or a plurality of filoviruses. As illustrated in the non-limiting Examples, the methods may include at least three, at least four, at least five or at least six different amino acid sequences from filovirus proteins, or fragments thereof from different filoviruses (e.g., NP, VP40, and GP-mucin, GP-ectodomain, etc. or fragments thereof).

In some embodiments, the methods can include amino acid sequences that are not identical to the protein sequence(s) from which the sequences are derived. Some minor changes in the primary amino acid sequence and/or post-translational modification and processing of the sequence may be included as long as the sequence change or modification does not interfere with the ability of the filovirus-specific antibody to bind the sequence. In some embodiments, the amino acid sequences have at least 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) to the filovirus protein from which the sequence(s) are derived. In some embodiments, the amino acid sequences may comprise an NP sequence having at least 90% sequence identity to the sequence selected from the group consisting of SEQ ID NO:4 (Zaire NP); SEQ ID NO: 10 (Sudan NP); SEQ ID NO: 16 (Bundibugyo NP); SEQ ID NO: 22 (Taï Forest NP); SEQ ID NO: 28 (Reston NP); and SEQ ID NO: 34 (Marburg NP); a VP40 sequence having at least 90% sequence identity to the sequence selected from the group consisting of SEQ ID NO: 2 (Zaire VP40); SEQ ID NO: 8 (Sudan VP40); SEQ ID NO: 14 (Bundibugyo VP40); SEQ ID NO: 20 (Taï Forest VP40); SEQ ID NO: 26 (Reston VP40); and SEQ ID NO: 32 (Marburg VP40); and/or a GP-mucin domain having at least 90% sequence identity to the sequence selected from the group consisting SEQ ID NO: 6 (Zaire GP-mucin); SEQ ID NO: 12 (Sudan GP-mucin); SEQ ID NO: 18 (Bundibugyo GP-mucin); SEQ ID NO: 24 (Taï Forest GP-mucin); SEQ ID NO: 30 (Reston GP-mucin); and SEQ ID NO: 36 (Marburg GP-mucin). In further embodiments, the methods may comprise an NP sequence selected from the group consisting of SEQ ID NO: 4 (Zaire NP); SEQ ID NO: 10 (Sudan NP); SEQ ID NO: 16 (Bundibugyo NP); SEQ ID NO: 22 (Taï Forest NP); SEQ ID NO: 28 (Reston NP); and SEQ ID NO: 34(Marburg NP); a VP40 selected from the list consisting of SEQ ID NO: 2 (Zaire VP40); SEQ ID NO: 8 (Sudan VP40); SEQ ID NO: 14 (Bundibugyo VP40); SEQ ID 20 (Taï Forest VP40); SEQ ID NO: 26 (Reston VP40); SEQ ID NO: 32 (Marburg VP40); and/or a GP-mucin domain selected from the group consisting of SEQ ID NO: 6 (Zaire GP-mucin); SEQ ID NO: 12 (Sudan GP-mucin); SEQ ID NO: 18 (Bundibugyo GP-mucin); SEQ ID NO: 24 (Taï Forest GP-mucin); SEQ ID NO: 30 (Reston GP-mucin); and SEQ ID NO: 36 (Marburg GP-mucin). In certain embodiments of the methods, the methods comprise the detection agent as described herein.

In some embodiments, the methods include incubation of the sample with a detection agent at temperature and for a period of time. While the time and temperature of the incubation, or reaction, may vary it will suitably fall within a range that allows for the specific binding of a filovirus specific antibody to an amino acid that it can bind, or in some embodiments, bind specifically. A further embodiment provides for temperatures and times that are effective to facilitate binding of an antibody in the sample to a filovirus protein or a fragment thereof, while avoiding nonspecific interaction between the antibodies and a filovirus protein or a fragment thereof.

In embodiments of the above methods, the sample or biological sample may comprise any biologically-derived material that may contain antibodies, or in which antibodies are typically present. In some embodiments, the sample may be derived from a mammal having a functioning or compromised an immune system. In some embodiments, the biological samples may comprise tissue, cells, or a biological fluid, such as blood (including serum, or whole blood obtained from a finger prick), GCF, amniotic fluid, BALF, salvia, tears, urine, lymphatic fluid, sputum, or cerebrospinal fluid taken from a mammal. In other embodiments, the biological sample may comprise cell cultures, cell lysates, or cellular fluids. In particular embodiments, the sample may comprise blood or serum.

In one embodiment of the aspects relating to methods, the methods can be used to detect and determine the presence of a filovirus specific antibody in a sample. In further embodiments, a method may comprise determining the amount of a filovirus specific antibody or a complex between a filovirus specific antibody and one or more filovirus antigens, proteins, or fragments thereof. Thus, the amount may be measured by determining the amount of the antibodies and/or complexes in a sample. Detection may be performed by any method and technique routinely used in the art such as, for example, using an antibody which is detectably labeled, or which can be subsequently labeled. A variety of formats can be employed to determine whether a sample contains a filovirus specific antibody. Immunoassay methods useful in the method detection can include, but are not limited to, dot blotting, western blotting, protein chips, immunoprecipitation (IP), competitive and non-competitive protein binding assays, enzyme-linked immunosorbant assays (ELISA), and others commonly used and widely-described in scientific and patent literature, and many employed commercially. One of skill in the art can readily adapt known protein/antibody detection methods for use in determining whether samples contain a biomarker (e.g., a filovirus specific antibody) and the relative concentration in the sample. Further, the methods can include processing of the sample to isolate a target (e.g., a filovirus specific antibody or complex thereof) using known techniques including, for example, those described in Harlow & Lane, Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1988).

Detection antibodies can be used in the methods and kits disclosed herein, including, for example, western blots or ELISA, to detect the formation of complexes formed between one or more filovirus-specific antibody and an amino acid sequence of a filovirus protein or fragment thereof. In such uses, it is possible to immobilize either the antibody or complexes on a solid support. Supports or carriers include any support capable of binding an antigen or an antibody, and are generally known in the art. Such supports may include, for example glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, ceramics, semiconductors, metal, and magnetite.

In one embodiment, the concentration of an antibody and/or one or more filovirus proteins or fragments thereof is determined in a biological sample obtained from a subject, including, for example, a human patient. For example, the antibody and/or the filovirus antigen can be isolated or purified from a sample obtained from cells, serum, tissue, or an organ of the subject, as discussed herein, and the amount thereof is determined. In some embodiments, the filovirus antibody and/or a complex comprising the filovirus antibody and an amino acid sequence of a filovirus protein or fragment thereof complex can be prepared from cells, tissue or organ samples by coimmunoprecipitation using an antibody immunoreactive with an interacting protein member, a bifunctional antibody that is immunoreactive with two or more interacting protein members of the protein complex, or an antibody selectively immunoreactive with the antibody and/or the complex. In some embodiments, bifunctional antibodies or antibodies immunoreactive with only free interacting filovirus antibodies are used, individual filovirus antibodies not complexed with other proteins may also be isolated along with the protein complex containing such individual antibodies. The complexes, filovirus specific antibodies and filovirus antigens may be separated from other proteins and biological materials/molecules in samples using methods known in the art, e.g., size-based separation methods such as gel filtration, or by removing the complex from the sample using another antibody having specific binding activity for the complex, filovirus antibody, and/or filovirus protein. Additionally, antibodies and proteins (and complexes between them) in a sample can be separated in a gel such as polyacrylamide gel and subsequently immunoblotted using an antibody immunoreactive with the protein and/or complex.

Alternatively, the concentration can be determined in a sample without separation, isolation or purification. For this purpose, an antibody selectively immunoreactive with the filovirus-specific antibody, filovirus antigen, and/or complex may be used in an immunoassay. For example, immunocytochemical methods can be used. Other antibody-based techniques are suitable and are generally known in the art including, for example, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoradiometric assays (IRMA), fluorescent immunoassays, protein A immunoassays, and immunoenzymatic assays (IEMA).

Methods of Manufacture

The disclosure also provides a method for making the detection agent as described herein comprising: expressing one or more recombinant polynucleotide sequences encoding an amino acid sequence of a filovirus protein, or a fragment thereof in an expression system; and fixing the encoded amino sequence of a filovirus protein, or a fragment thereof, on a surface of the substrate. The expression system includes a prokaryotic cell, a eukaryotic cell, or in vitro translation, or any combination thereof. The prokaryotic cell comprises E. coli. The eukaryotic cell is selected from the group consisting of yeast, an insect cell, and a mammalian cell.

In these embodiments, the methods further comprise the general techniques and reagents that are known in the art and which may find common use in preparing detection agents (e.g., protein microarrays, protein-based microparticles, and/or protein-based nanoparticles).

Kits

Another aspect relates to a kit including a detection agent as disclosed herein, at least one reagent that can detect a filovirus-specific antibody bound to the detection agent, and instructions for use of the kit. A kit may be used for conducting the diagnostic and screening methods described herein. Typically, the kit should contain, in a carrier or compartmentalized container, and additional reagents and buffers useful in any of the above-described embodiments of the diagnosis method. The carrier can be a container or support, in the form of, for example, bag, box, tube, rack, and is optionally compartmentalized. The carrier may define an enclosed confinement for safety purposes during shipment and storage. In one embodiment, the kit includes an antibody that is selectively immunoreactive with a complex comprising a filovirus-specific antibody and an amino acid sequence of a filovirus protein, or fragment thereof as described herein. In some embodiments the kit includes an antibody that is selectively immunoreactive with a filovirus-specific antibody (e.g., an anti-mammal antibody, such as an anti-human antibody) that can detect the presence of the filovirus-specific antibody bound to the detection agent. The antibodies may be labeled with a detectable marker such as radioactive isotopes, or enzymatic or fluorescence markers. Alternatively, additional secondary antibodies such as labeled anti-IgG and the like may be included for detection purposes. Optionally, the kit can include one or more of proteins sequences as a control or comparison purposes. Instructions for using the kit or reagents contained therein are also included in the kit.

The above aspects and embodiments are further illustrated in the non-limiting Examples that follow. While the Examples may refer to specific aspects of the disclosure, it will be appreciated that the information is merely provided for purposes of illustration and exemplification of the broader disclosure.

EXAMPLE 1

Cloning. Full-length genes for NP and VP40, and the GP mucin-like domain fragment (GP-mucin) for six filovirus species: Reston (REBOV), Bundibugyo (BEBOV), Zaire (ZEBOV), Sudan (SEBOV), and Taï Forest ebolavirus (TAFV); and Marburg marburgvirus (MARV) were cloned into pENTR™/TEV/D-TOPO® vector (Life Technologies, Grand Island, N.Y.) and sequence-verified. The nucleotide substitutions found in cloned sequence compared with the reference sequence from GenBank are summarized in Table 1. All entry vector clones were shuttled into destination E. coli expression vectors via LR reaction (LR Clonase® II, Life Technologies). Specifically, VP40 (Zaire VP40—SEQ ID NO: 1; Sudan VP40—SEQ ID NO: 7; Bundibugyyo VP40—SEQ ID NO: 13; Taï Forest VP40—SEQ ID NO: 19; Reston VP40—SEQ ID NO: 25; and Marburg VP 40—SEQ ID NO: 31) and GP-mucin (Zaire GP-mucin—SEQ ID NO: 5; Sudan GP-mucin—SEQ ID NO: 11; Bundibugyo GP-mucin—SEQ ID NO: 17; Taï Forest GP-mucin—SEQ ID NO: 23; Reston GP-mucin—SEQ ID NO: 29; and Marburg GP-mucin—SEQ ID NO: 35) ORFs were shuttled into pDESTHisMBP (Addgene plasmid 11085) containing an N-terminal HisMBP tag, while all NP (Zaire NP—SEQ ID NO: 3; Sudan NP—SEQ ID NO: 9; Bundibugyo NP—SEQ ID NO: 15; Taë Forest NP—SEQ ID NO: 21; Reston NP—SEQ ID NO: 27; and Marburg NP—SEQ ID NO: 33) ORFs were shuttled into pDEST17 (Life Technologies) containing an N-terminal His tag.

TABLE 1 Summary of cloned filovirus sequences compared to GenBank reference sequences Amino Acid GenBank Nucleotide Species¹ Gene Residues Sequence Substitutions³ Bundibugyo VP40 1-326 FJ217161.1 None ebolavirus NP 1-739 FJ217161.1 C1735T (silent) GP-mucin 313-465  FJ217161.1 C6973T (silent), A7363G (silent) Tai Forest VP40 1-326 FJ217162.1 None ebolavirus NP 1-739 FJ217162.1 None GP-mucin 313-465  FJ217162.1 None Reston VP40 1-331 AF522874.1 G4490A (silent), A5466G (Asn to Asp) ebolavirus NP 1-739 AF522874.1 T2188C (silent) (Pennsylvania) GP-mucin 314-466  AY769362. G7093A (silent) Sudan VP40 1-326 FJ968794.1 T4465C (silent) ebolavirus NP 1-738 AF173836.1 C2581T (silent) (Boniface) GP-mucin 313-465  FJ968794.1 A7112G (silent) Zaire VP40 1-326 AF499101.1 G4496A (silent), A4592G (silent), ebolavirus T5204C (silent) (Mayinga) NP 1-739 AF086833.2 A491G (Ile to Val) GP-mucin² 313-465  JQ352783.1 None Marburg VP40 1-303 DQ217792.1 None marburgvirus NP 1-695 DQ217792.1 None (Musoke) GP-mucin 289-505  DQ217792.1 A6906T (silent) ¹Where available, strain names are in parentheses. ²Zaire ebolavirus GP-mucin sequence is from the Kikwit strain. ³The position of nucleotide substitutions are based on GenBank sequence as reference. Corresponding amino acid change is noted in parentheses. Otherwise, the mutation is noted as silent.

Protein expression and purification. Proteins VP40 (Zaire VP40—SEQ ID NO: 2; Sudan VP40: SEQ ID NO: 8; Bundibugyo VP40—SEQ ID NO: 14; Taï Forest VP40—SEQ ID NO: 20; Reston VP40—SEQ ID NO: 26; and Marburg VP40—SEQ ID NO: 32), NP (Zaire NP—SEQ ID NO: 4; Sudan NP—SEQ ID NO: 10; Bundibugyo NP—SEQ ID NO: 16; Taï Forest NP—SEQ ID NO: 22; Reston NP—SEQ ID NO: 28; and Marburg NP—SEQ ID NO: 34), GP-mucin (Zaire GP-mucin—SEQ ID NO: 6; Sudan GP-mucin—SEQ ID NO: 12; Bundibugyo GP-mucin—SEQ ID NO: 18; Taï Forest GP-mucin—SEQ ID NO: 24; Reston GP-mucin—SEQ ID NO: 30; and Marburg GP-mucin—SEQ ID NO: 36) were expressed in either BL21-AI™ cells (Life Technologies) or Rosetta™ 2(DE3) cells (EMD Millipore, Billerica, Mass.). Expression for pDESTHisMBP constructs was induced with 1 mM IPTG, while expression for pDEST17 18° C. was induced lysed using with 0.2% L-arabinose. Pelleted cells from overnight cultures grown at 18° C. were lysed using B-PER reagent (Thermo Scientific, Rockford, Ill.) supplemented with 2× HaIt™ Protease and Phosphatase Inhibitors Cocktail, EDTA-free (Thermo Scientific); 0.2 mg/mL lysozyme; 50-100 U/mL DNasel (Thermo Scientific); and 2 mM PMSF. Lysates were separated into supernatant and insoluble pellet fractions by centrifugation, and induced protein expression was confirmed through Western blotting or mass spectrometry and Coomassie staining. HisMBP-tagged VP40s and GP-mucins were soluble and present in the supernatant fraction. With the exception of ZEBOV NP (Zaire-NP) (SEQ ID NO: 4), all His-tagged NPs were insoluble and predominantly in the pellet fraction. Supernatant containing expressed VP40s were loaded onto HisTrap™ HP columns (GE Healthcare, Piscataway, N.J.) pre-equilibrated with 20 mM sodium phosphate, 0.5 M NaCl, 40 mM imidazole, pH 7.4. VP40 fractions were collected by applying an imidazole step elution. All GP-mucins except MARV GP-mucin (Marburg-GP-mucin) were purified using HisTrap™ HP columns. Binding and washing steps were conducted with 25 mM HEPES, 0.5 M NaCl, 25 mM imidazole, pH 8, and bound GP-mucins were eluted using an imidazole gradient. Marburg-GP-mucin was purified using MBPTrap™ HP column pre-equilibrated with 25 mM HEPES, 0.2 M NaCl, 1 mM EDTA, pH 7.4. Bound protein was eluted using 25 mM HEPES, 0.2 M NaCl, 1 mM EDTA, 10 mM maltose, pH 7.4. NPs were purified through on-column refolding on HisTrap™ HP columns. Briefly, NP pellets were re-solubilized in 25 mM HEPES, 0.2 M NaCl, 25 mM imidazole, 1 mM beta-mercaptoethanol, 6 M guanidine hydrochloride, pH 8. Proteins were bound to columns under denaturing conditions and refolded using a 6 to 0 M urea gradient over a 30 column volume range. Refolded proteins were eluted using an imidazole gradient. Although Zaire-NP was found in the supernatant, the protein did not appear to bind to the HisTrap™ column under the conditions used for VP40 purification. This may have been due to a hidden His tag, and thus, guanidine hydrochloride was added directly to Zaire-NP supernatant to a final concentration of 6 M in order to expose the His tag. Denatured Zaire-NP was processed in a similar manner as the other re-solubilized NPs. Purity and concentration of collected fractions were measured by Agilent Protein 230 kit (Agilent Technologies). All purified proteins were stored at −20° C. in their respective elution buffers with glycerol added to a final concentration of 25%.

Microarray printing. The purified recombinant proteins were spotted (FIG. 4A-B) on nitrocellulose-coated FAST® slides (KeraFAST, Boston, Mass.), using a contactless inkjet microarray printer (ArrayJet, Edinburgh, Scotland). The microarray included a total of 34 proteins: i) E. coli-expressed filovirus antigens; ii) insect cell-expressed ZEBOV and SEBOV and Marburg virus Angola) GP ectodomain (ΔTM) (IBT Bioservices, Gaithersburg, Md.); iii) mammalian cell-expressed Marburg virus (Musoke) GP ΔTM (IBT Bioservices); iv) human, monkey, mouse, rabbit, and goat IgG (Rockland Immunochemicals, Gilbertsville, Pa.); v) human, monkey, and rabbit IgM (Rockland Immunochemicals; vi) HisMBP (ProteinOne, Rockville, Md.); vii) dengue virus serotype 2 (dengue2) and 3 (dengue3) nonstructural protein 1 (NS1); and viii) BSA (Thermo Scientific). The purified dengue virus proteins were previously described (36). Briefly, the proteins were expressed with a HisMBP tag in E. coli and purified via immobilized metal affinity chromatography. Each protein was printed in triplicates. All purified proteins were diluted to 200 ng/uL in printing buffer (25 mM HEPES, 0.5 M NaCl, 25% glycerol, 1 mM DTT, pH 8). Alexa Fluor® 647-conjugated streptavidin (Life Technologies) was diluted 1:50 in printing buffer and included in the microarray as reference markers. Buffer served as empty placeholders on the microarray. Printed slides were desiccated overnight under vacuum and stored at −20° C.

Microarray processing. All microarray processing steps were performed under 21° C. conditions, and each antibody or serum sample was processed in duplicate microarrays. Printed microarrays were incubated for 1 hour in 1× Biacore Flexchip blocking buffer (GE Healthcare) with 2% normal goat serum (Vector Laboratories, Burlingame, Calif.) or 2% normal rabbit serum. Microarrays were washed three (3) times at five (5) minutes each with wash buffer (1× TBS, 0.2% Tween 20, 3% BSA) which was used in all subsequent wash steps. Microarrays were incubated with primary antibody diluted 1:1000 or serum sample diluted 1:150 in probe buffer (1× TBS, 0.1% Tween 20, 3% BSA). After 1 hour incubation in primary antibody or sera, microarrays were washed and incubated 1 hour with Alexa Fluor® 647-conjugated secondary antibodies diluted 1:2000 in probe buffer. Microarrays were washed, and then rinsed with water before analysis.

Vaccinations and infections. Rhesus macaque sera were obtained from two separate vaccine studies for ZEBOV and MARV. The vaccine trials were similar in design and procedure to a study previously described by Warfield et al. (37). Briefly, for the ZEBOV study, five animals were vaccinated with ZEBOV virus-like particles (VLP) and MARV VLP. The vaccinated animals were subsequently challenged with ZEBOV. Three sera were collected for each animal: naïve, post-immunization, and post-challenge. TheMarburg virus study was conducted in a similar manner, except that the animals were vaccinated with MARV VLP and challenged with MARV.

Antibodies. Rabbit polyclonal anti-ZEBOV NP (anti-Zaire-NP, 0301-012), mouse monoclonal anti-SEBOV GP (anti-Sudan-GP, 0202-029), mouse monoclonal anti-SEBOV VP40 (anti-Sudan-VP40, 0202-018), mouse monoclonal anti-ZEBOV GP (anti-Zaire-GP, 0201-020), rabbit polyclonal anti-ZEBOV VP40 (anti-Zaire-VP40, 0301-010), mouse monoclonal anti-Marburg virus (Musoke) GP (anti-Marburg-GP, 0203-023), and mouse monoclonal anti-Marburg virus (Musoke) VP40 (anti-Marburg-VP40, 0203-012) antibodies were purchased from IBT Bioservices. Alexa Fluor® 647-conjugated goat anti-mouse IgG (A21237) and goat anti-rabbit IgG (A21244) antibodies were purchased from Life Technologies. Alexa Fluor® 647-conjugated rabbit anti-monkey IgG (bs-00335R-A647) and rabbit anti-monkey IgM (bs-0336R-A647) antibodies were purchased from Bioss (Woburn, Mass.).

Data acquisition and analysis. Processed slides were scanned at 635 nm wavelength using GenePix® 4400A (Molecular Devices, Sunnyvale, Calif.). Acquired images were analyzed with GenePix Pro 7 software. Any defective or missing spots were removed from further analysis. Median fluorescence intensity for each microarray spot was corrected through local background subtraction on GenePix Pro 7. Subsequent analysis was done in Microsoft Excel and R. The resulting background-corrected fluorescence intensities were averaged across replicate spots and quantile-normalized for each serum group (naïve, immunized, and challenged). A paired t-test was conducted to compare each antigen-antibody signals for naïve versus immunized, naïve versus challenged, and immunized versus challenged sera.

RESULTS

Filovirus protein microarray. Taking into consideration the complexity of the viral proteome and previous data suggesting potential targets of host antibody responses (14-16, 38), we developed a microarray comprised of a minimal set of proteins representative of all Marburg and Ebola virus species. The VP40 and NP for Reston, Bundibugyo, Zaire, Sudan, and Taï Forest ebolovirus and Marburg marburgvirus MARV were expressed as full-length recombinant proteins in E. coli. Initially, we prepared GP ectodomains (ΔTM) constructs from all filovirus species for expression in E. coli. However, because the GP ΔTM proteins were not all stable in solution, the coding sequences were truncated and expressed as more stable, GP mucin-like domain fragments (Table 1), with HisMBP fusion tags (amino-termini). The final GP protein design was supported by data from previous reports suggesting that antibody responses to ZEBOV were directed at least in part against the GP mucin-like domain (39-42).

The recombinant filovirus antigens purified from E. coli, along with control proteins, were printed in 120-130 micron diameter spots in a 12×12 format (FIG. 4) on slides covered with a thin layer of nitrocellulose. Additionally, GP ΔTM produced in eukaryotic host cells were included in the microarray for comparison with the E. coli-produced GP-mucins. IgGs (monkey, human, rabbit goat, and mouse), IgMs (human, monkey, and rabbit), HisMBP, BSA, and dengue virus proteins served as controls. For quality control purposes and to validate our assay design, printed microarrays were probed with anti-His antibody as well as a panel of purified filovirus antibodies. Probing with anti-His antibody showed that all His-tagged proteins were successfully spotted and adsorbed onto the nitrocellulose-coated microarray slides (data not shown). Anti-Marburg-VP40, anti-Marburg-GP, anti-Sudan-VP40, anti-Sudan-GP, anti-Zaire-VP40, anti-Zaire-NP, and anti-Zaire-GP were bound by their target antigens with a high degree of specificity (FIG. 1A, B, C). Minor cross-reactivity between REBOV-VP40 and Sudan-VP40, and between BEBOV-VP40 and ZEBOV-VP40 were observed when microarrays were probed with anti-SEBOV-VP40 and anti-ZEBOV-VP40, respectively (FIG. 1B, C). Combined, data from these control antibodies indicate that the filovirus microarrays performed correctly under idealized test conditions.

Analysis of sera from ZEBOV and MARV challenge studies. Sera from two separate animal studies were analyzed using our microarrays. In the ZEBOV study, rhesus macaques were vaccinated with a mixture of trivalent (GP, NP, and VP40) virus-like particles (VLP) for MARV and F and subsequently challenged with ZEBOV. In the Marburg virus study, rhesus macaques were vaccinated with trivalent (GP, NP, and VP40) VLP for MARV and subsequently challenged with MARV. All vaccinated animals in the Zaire and Marburg studies survived the viral challenge. After applying the serum samples to the filovirus microarray, bound IgG were detected using fluorescently-labeled secondary antibodies (FIGS. 5 and 6).

For the ZEBOV study, comparison between sera from naïve and immunized animals showed significant increases (p<0.05) in IgG against all vaccine antigens except for MARV NP (Marburg-NP) (FIG. 2A). Cross-reactive IgG against BEBOV, TAFV, REBOV, and SEBOV VP40, and BEBOV and TAFV NP were induced through vaccination (FIG. 2A). After animals were challenged, IgG signals against all Ebola virus NPs and VP40s and ZEBOV GP-mucin had significant increases (p<0.005) in challenged sera compared to immunized sera (FIG. 2A). For the Marburg virus study, the microarrays detected significant increases (p<0.05) in IgG against Marburg-NP, -GP-mucin, and -VP40 in immunized sera compared to naïve sera (FIG. 2B). Cross-reactive IgG against all Ebola virus VP40s were detected in the immunized sera (FIG. 2B). We observed a cross-reactive signal against Zaire-GP-mucin which was statistically significant (p<0.05) comparing naïve and immunized sera but not between naïve and challenged sera (FIG. 2B). Comparison between naïve and challenged sera showed significant increases (p<0.05) in IgG responses for Marburg-NP and -GP-mucin (FIG. 2B). However, the increase in IgG against Marburg-VP40 was not statistically significant (FIG. 2B). The results from analysis of rhesus sera suggested that the microarray enabled detection of anti-GP antibodies in a species-specific manner. Further, the anti-GP antibodies were detected with minimal cross-reactivity towards other species for the case of sera from the ZEBOV and MARV infections (FIG. 2C). We also examined IgM responses with sera from both animal studies, and representative data are provided in Supplementary FIG. 4. Overall, minor IgM signals were detected against ZEBOV and MARV antigens using these convalescent sera. The preliminary results indicate that the filovirus microarray may be used for IgM detection. Analysis of sera collected from time points closer to vaccination and viral challenge will confirm the utility of measuring IgM responses by protein microarray.

Comparison between E. coli and eukaryotic cell-expressed GP. Both the GP-mucins produced in E. coli and GP ΔTMs produced in eukaryotic cells (insect or mammalian) were included in the printed microarray. Examining sera from the ZEBOV (FIG. 3A) and MARV (FIG. 3B) studies, we confirmed that the mucin domain was sufficient for capturing IgG responses to filoviruses. We observed slightly higher IgG signals from the Zaire GP-mucin compared to Zaire-GP ΔTM with sera from ZEBOV challenged animals (FIG. 3A, B), whereas antibody recognition of the Marburg GP-mucin was comparable to the GP ΔTM Marburg (both Angola and Musoke) for sera obtained from animals challenged with MARV. Based on these microarray results, we concluded that the E. coli-produced GP-mucin resulted in similar species-specificity as the eukaryotic cell-expressed GP ΔTMs.

DISCUSSION

As the above example and data show, the compositions and methods disclosed herein, as demonstrated through one protein microarray embodiment provide for a platform that can identify and examine the antibody responses of mammals (e.g., rhesus macaques) to infection and vaccination (e.g., various species of Ebola and Marburg viruses). The illustrative florescence-based readout for the microarray shows that the assay and methods are highly sensitive, and only requires a minimal volume (1-2 microliters) of sample in order to provide a complete evaluation. While any number of amino acid sequences from one or more filovirus proteins may be used in connection with the detection agent and methods, the NP and GP antigens were very sensitive and could distinguish sera from ZEBOV in comparison to Marburg virus infection. The results from the Marburg virus study sera (Marburg-VP40) showed that some amino acid sequences can be associated with an amount of cross-reactivity to the VP40 antibody response against all Ebola viruses. Similarly, the results shown herein also were able to identify a general antibody cross-reactivity among Ebola virus NP and VP40 proteins, which is similar to results from previously reported ELISA studies (43-45). The data also identified that under these assay conditions, GP exhibited the highest level of antibody specificity. Further, and supporting the relevance of the GP-mucin domain as a serological marker of infection, E. coli-expressed GP-mucins for Zaire and Marburg filoviruses displayed similar species-specific antibody recognition as the multi-domain GPs (ΔTM) that were produced from eukaryotic cells, based on assay results from the ZEBOV and MARV studies. The microarray assay detected increases in IgG responses to specific filovirus antigens resulting from vaccination or viral challenge, and the relative levels of other antibody isotypes (IgM) could also be measured. We further noted that active infection stimulated a significant boost in immune responses primed by vaccination, as specific IgG levels in VLP-vaccinated macaques increased in response to aerosol challenges from either ZEBOV or MARV. The significant increase in ZEBOV and MARV-specific IgG following viral challenge, as measured by the protein microarray, shows that VLP vaccinations did not induce sterilizing immunity in the animals.

The results corroborate previously reported studies concerning antibody recognition of filovirus antigens. Antibody responses against NP and GP are detected in human patients by ELISA (14-16) and Western blots (46). Other reports have observed antibodies that recognize GP, NP, and VP40 in sera from a SEBOV (Gulu) outbreak in 2000-2001(38). However, these previous ELISA studies examined only select antigens from a single filovirus species or a single antigen from multiple filoviruses, whereas the microarray format supports a highly multiplex analysis of sera. By providing these Examples which demonstrate that more than one species of filovirus (e.g., two species of virus) can be examined and identified using the techniques and compositions disclosed herein, the disclosure provides for detection agents and methods (e.g., protein microarray) useful for multiplexed study of serological responses to most filovirus strains. The disclosure expands the capabilities of any previously described methods and compositions and facilitates diagnosis and serological surveillance of infections caused by multiple species of the highly infectious filoviruses. Further, providing for detection agents and methods that can be adapted into a low-cost, point-of-care assay greatly extends the utility of the technology, (e.g., relative to the prior methods and techniques requiring full laboratory facilities).

Management and patient care for typical filoviral infections provide significant challenges given the usually resource-poor settings of outbreaks and the procedures that are required to prevent spread of infections (47). Allaranga and coworkers proposed that an active epidemiological surveillance system, including surveillance of zoonotic infections, is vital for early detection and effective response to filoviral hemorrhagic fever epidemics in Africa (48). A recent report of hospital-based surveillance in Ghana identifies the importance of distinguishing infections caused by hepatitis viruses that produce symptoms that mimic viral hemorrhagic fevers from the infrequent infections caused by filoviruses (49). Further, the prevailing hypothesis concerning outbreaks of filoviral hemorrhagic fevers is that indigenous human populations occasionally make contact with animal reservoirs of Ebola and Marburg viruses, resulting in rapid spread of disease (Mbonye et al, 2013). Wildlife are often more severely affected than humans, as demonstrated by a 89% drop in chimpanzees and 50% decrease in gorilla populations as a result of one recorded Ebola virus outbreak (50). Thus, the disclosure provides compositions and assay methods that can be incorporated as a vital tool for such epidemiological studies and for eventual diagnosis of infections, including supporting serological surveillance of infections occurring within domestic or wildlife animal populations.

EXAMPLE 2

This example illustrates the specificity of antibody responses with sera collected from survivors of three separate Ugandan outbreaks that were caused by Marburg marburgvirus (MARV) in Kabale, Bundibugyo ebolavirus, (BDBV) in Bundibugyo, and Sudan ebolavirus (SUDV) in the Gulu district. Control samples collected from the same geographical regions as the disease outbreaks were also included in the study. To measure antibody responses, we assembled a protein microarray that displayed nucleoprotein (NP), varion protein 40 (VP40), and glycoprotein (GP) antigens from isolates representing the six species of filoviruses. Analysis of the microarray data by hierarchical clustering revealed clear positive signals from all infection samples, which were readily distinguishable from negative controls. The amino acid sequences of GP are most diverse among species, whereas NP sequences are highly conserved. Consistent with protein similarities, NP was most cross-reactive and exhibited the highest level of antibody responses, while antibody responses to GP were the most specific. Persistent antibody levels to GP, NP and VP40 were observed for Gulu SUDV survivors 14 years after infection. Significant antibody responses to autologous antigens were observed for all three outbreak cohorts. The MARV survivors presented the lowest level of antibody cross-reactivity with proteins from heterologous filoviruses, while the SUDV survivors exhibited the highest cross-reactivity with other filoviral proteins. Our results suggest that survival from infection caused by one species of filovirus may impart at least partial immunity to other outbreaks.

Methods:

Ebola and Marburg Survivor Sera

Our study included a total of 59 serum samples from patients who survived infections caused by SUDV-Gulu, BDBV-Bundibugyo and MARV-Kabale outbreaks along with controls from subjects living in the same location who were not infected with the virus. The survivors received uniform treatment after admission to a hospital. Institutional approvals for the study were obtained from the Uganda Virus Research Institute in Entebbe, Uganda, Ugandan National Council for Science and Technology and United States Army Medical Research Institute of Infectious Diseases (USAMRIID). A signed consent form and a personal health questionnaire were obtained from each subject. A serum sample from a human subject who was vaccinated with a recombinant adenovirus serotype 5 (rAd5) expressing EBOV and SUDV GP was also included in the study (Ledgerwood, Costner et al. 2010).

Sequence and Phyologeny Analysis

Three multiple sequence alignments (MSAs) were generated for the amino acid sequences of NP and GP mucin-like domains of Ebolavirus and Marburgvirus strains, using CLUSTAL W2 (Larkin, Blackshields et al. 2007). Each MSA had a different gap opening penalty (5, 10, and 25), with Blosum62 as the protein weight matrix and all other options left as default. T-Coffee Combine (Notredame, Higgins et al. 2000, Di Tommaso, Moretti et al. 2011) was then used to generate a single alignment that had the best agreement of all three MSAs for each protein. The combined alignments of full-length NP and GP mucin-like domain sequences were used to calculate Shannon entropy per column of the aligned sequences as a measure of amino acid variability and to generate percent identity matrices in BioEdit Sequence Alignment Editor v7.1.3.0 (Hall 1999). To eliminate poorly aligned positions and divergent regions in the combined alignments, each alignment was filtered using Gblocks (Castresana 2000, Talavera and Castresana 2007) with strict settings (no gap positions within the final blocks, strict flanking positions, and no small final blocks). Gblocks identified a 406 residue conserved region at the N-terminus of NP, which was used for phylogenic reconstruction (BDBV, TAFV, RESTV, SUDV, EBOV-residues 20-425; MARV-residues 2-407). Due to a high degree of heterogeneity among individual residues, conserved regions within GP mucin-like domain sequences could not be identified. For this reason, an ungapped, highly variable region of 33 residues at the N-terminal portion of the GP moiety was selected for use in phylogenic reconstruction (BDBV, TAFV, RESTV-residues 2-34; EBOV and MARV-residues 1-33). Phylogenic trees were generated using the maximum likelihood method implemented in the PhyML program (v3.0 aLRT) (Guindon, Dufayard et al. 2010). The Blosum62 substitution model was selected and 4 gamma-distributed rate categories to account for rate heterogeneity across sites. The gamma shape parameter was estimated directly from the data (α_(NP)=0.654, α_(GPmucin)=15.371). Tree topology and branch length were optimized for the starting tree with subtree pruning and regrafting (SPR) selected for tree improvement. Reliability for internal branches was assessed using a bootstrap method with 1000 replicates. Comparison of phylogenetic trees was completed using Compare2Trees software available online (Nye, Lio et al. 2006).

Microarray Proteins

Recombinant proteins from filoviruses were cloned, and the GP-mucin, NP and VP40 were expressed in E. coli, while the GPΔTM proteins were produced in insect or mammalian expression systems as previously described (Kamata, Natesan et al. 2014). The GPΔTM for BDBV, SUDV (Boniface), Zaire Ebola virus (EBOV-Mayinga), Reston Ebola virus (RESTV-Reston), and MARV (Angola) were produced in insect cells; while BDBV, RESTV (Pennsylvania), Tai Forest Ebola virus (TAFV), SUDV (Boniface), EBOV (Mayinga), and MARV (Musoke) GPΔTM were expressed in mammalian cells. The Ebola GP-mucins were purified using HisTrap HP columns and MARV GP-mucin was purified using MBPTrap HP column. The NPs were purified by on-column refolding on HisTrap HP columns as described previously (Kamata, Natesan et al. 2014). The purity and concentrations of the proteins were determined by microfluidic assays (Agilent Technologies, Santa Clara, Calif.). The dengue virus proteins were previously described (Fernandez, Cisney et al. 2011).

Multiplexed Protein Microarray

The recombinant proteins were spotted (140 μm diameter) in a 10×36 microarray on FAST® slides (Kerafast, Boston, Mass.) by using a Marathon inkjet microarrayer (ArrayJet, Edinburgh, Scotland, UK). The array included a total of 41 proteins: i) E. coli-expressed filoviral antigens; ii) Sf9-expressed GPΔTM from MARV, EBOV, SUDV, BDBV and RESTV (IBT Bioservices); iii) mammalian cell-expressed GPΔTM from from all six species of Filoviridae iv) human, monkey, mouse, rabbit, and goat IgG (Rockland Immunochemicals, Gilbertsville, Pa.); v) HisMBP (ProteinOne, Rockville, Md.); iv) human, monkey, and rabbit IgM (Rockland Immunochemicals) and vi) dengue virus serotype 2, 3 nonstructural protein 1 (NS1); and vi) BSA (Thermo Fisher Scientific, Grand Island, N.Y.). The expression and purification of the dengue virus proteins was previously described (Fernandez, Cisney et al. 2011). Each protein in the microarray was printed in quadruplicate. All purified proteins were diluted to 200 ng/μL and prepared in a final concentration of 50% glycerol in printing buffer consisting of 25 mM HEPES, 0.5 M NaCl, and 1 mM dithiothreitol (DTT). Alexa647®-conjugated streptavidin (Life Technologies) was diluted 1:50 in 1× PBS with 50% glycerol and included in fixed positions within the array as a spatial reference marker. The recombinant proteins were physically characterized to confirm correct molecular weight and purity (70-95%; data not shown). Quality control of printed microarrays was confirmed with specific antibodies against poly-His tags, Marburg VP40, Marburg GP, Sudan VP40, Sudan GP, anti-Zaire VP40, Zaire NP and Zaire GP, as described previously (Kamata and Natesan, 2014). The printed and dried microarrays were stored under vacuum (−20° C.).

Analysis of Antibody Interactions

A Tecan HS Pro400 (Tecan US, Morrisville, N.C.) hybridization station was used for most of the microarray processing steps, and all manipulations were performed at 22° C. The printed microarray slides were incubated for 1 hour in blocking buffer (1× Biacore Flexchip, GE Healthcare) with 2% normal goat serum (Vector Laboratories). The microarray slides were washed (3 times; 5 minute each) with a buffer (1× TBS, 0.2% Tween 20, 3% BSA) that was used in all subsequent wash steps. Human sera diluted 1:150 in probe buffer (1× TBS) were incubated (1 hour) on the microarray surface. The slides were washed and incubated for 1 hour with Alexa®647-conjugated secondary antibodies diluted 1:2000 in probe buffer. The slides were rinsed with water and dried before acquiring data.

Data Acquisition and Analysis

Processed slides were scanned at 635 nm wavelength using a GenePix® 4400A (Molecular Devices, Sunnyvale, Calif.), with PMT gain set to 400 and laser power set to 10%. Acquired images were analyzed using GenPix Pro 7 software (Molecular Devices). Background median fluorescence intensity was subtracted from median fluorescence intensity of each spot, and the resulting background-corrected fluorescence intensity was averaged across the quadruplicates. The calculated mean fluorescence was used for further analysis. The data were quantile normalized using the preprocess core package of R. Background correction, standard Z-score normalization to compare each signal to that of all signals, and M—statistics were with performed with ProtoArray Prospector software (Life Technologies), as described previously (Keasey, Schmid et al. 2009). Group comparisons between control and survivors were performed with thresholds of normalized signals of at least 500 relative florescence units (RFUs), and a minimal signal difference of 200 RFU between two groups. Heat maps of normalized and log 2-transformed values were created using GENE-E (Broad Institute, Cambridge, Mass.). Hierarchical clustering by average linkage Euclidean distance was used to examine overall patterns of antibody interactions.

Results

Amino Acid Sequence Diversity of Filovirus Proteins

To examine human antibody responses to filovirus infections we first considered the selection of protein probes to include in our analysis. We performed a phylogenic analysis of filoviral proteins (FIG. 8) to identify highly conserved probes as well as proteins that may provide an antibody response signature that was unique to each species of virus. As demonstrated in FIG. 8, NP is highly conserved among ebolaviruses (>60% sequence identity), while NP from MARV shares only 30% sequence identity with ebolavirus species (FIG. 8c ). Approximately 400 amino acids of the N-terminal portion of NP that showed a higher degree of similarity among ebolavirus strains and MARV (Keasey, unpublished data; (Sanchez, Kiley et al. 1992), was selected for phylogenic inference (FIG. 8a ). The small shape parameter value (α_(NP)=0.654) of the gamma distribution used for construction of the dendrogram based on NP sequences indicated that there was a relatively large amount of rate variation, with many sites evolving very slowly and select sites evolving at a high rate (Lio and Goldman 1998). Thirty percent of NP residues (216 residues out of 739 total) were completely conserved among the six strains examined, with an average variability/residue=0.638 (data not shown), based on Shannon entropy calculations per residue of the NP MSA. In contrast, the mucin-like domain of GP exhibited minimal sequence identity among all filovirus strains examined (sequence identity=5-26%, 8). Phylogenic inference was based on only a 33 residue region at the N-terminus of the domain (FIG. 8b ), due to the fact that this was the only ungapped portion of the multiple sequence alignment. The shape parameter of the gamma distribution (α_(GPmucin)=15.371) for GP-mucin sequences was much larger than that of NP sequences, indicating that most sights have roughly similar rates of substitution (Lio and Goldman 1998). No residues within the mucin-like domain were completely conserved, and the average variability per residue was 50% greater than that of NP sequences (H/res=1.07). Further, the MARV GP mucin-like domain sequence is 225 residues in length versus 153 residues for ebolavirus strains.

We measured topological features among filovirus strains based on amino acid sequences of NP and GP-mucin (Compare2Trees software tool), (Nye, Lio et al. 2006). Comparison of phylogenic trees (FIG. 8 a, b) reveals a similar topology for NP and GP-mucin, despite extensive sequence diversity among individual proteins. The overall similarity between the two trees was found to be 77.8%, with BDBV, TAFV, EBOV edges being 100% conserved between trees, and SUDV, RESTV, and MARV edges exhibiting 66.7% similarity. The long branch length of MARV separated this lineage of filoviruses from all ebolavirus species.

Human Antibody Reponses to Filoviral Proteins

The study involved a total of 37 survivors from the 2000 SUDV-Gulu outbreak, 20 samples from 2007 BDBV-Bundibugyo outbreak, and 2 samples from MARV-Kabale outbreak (Table 2). The sera samples were collected from a year after outbreak for MARV-Kabale, seven years later for BDBV-Bundibugyo, and twelve to fourteen years after for SUDV-Gulu. Sera collected from non-infected individuals living in the same geographical region of each outbreak, normal healthy volunteers from the United States, and serum from a subject vaccinated with a replication defective rAd5 vaccine expressing GP antigens in a 1:1 ratio from EBOV of Zaire strain and SUDV of Gulu strain (Ledgerwood, Costner et al. 2010) were included as controls. To examine antibody interactions, dilutions of each serum were incubated on the surface of the protein microarray, which included eleven GPΔTM proteins produced by eukaryotic cell expression, eighteen proteins (GP-mucin, NP and VP40 from six species) expressed in E. coli (Table 3).

TABLE 2 Summary of sample used in the study Year of Year of No. of No. of Location Species Outbreak Collection Samples Controls Gulu SUDV 2000 2012/2014 37 4 (Uganda) Bundibugyo BDBV 2007 2014 20 3 (Uganda) Kabale MARV 2012 2013 2 — (Uganda) USE-NIAID EBOV — — 1 5 rAd5 vector SUDV vaccine (GP)

A heat map showing the analysis of all serological immune responses to the filovirus proteome is illustrated in FIG. 9. Hierarchical clustering (unsupervised) of the data by Euclidean distance indicated that the human sera was organized into two major clusters corresponding to infected and uninfected (negative) control groups (FIG. 9). Within the negative control group, the non-African, BDBV negative controls, and SUDV negative samples clustered separately (FIG. 9). The non-African control sera exhibited no antibody interactions with the filoviral proteins, while sera collected in Uganda showed some but not significant binding to filoviral proteins. The MARV, BDBV and SUDV convalescent samples clustered as distinct groups within the infected sample group (FIG. 9), indicating that the IgG signals from our microarrays can be used to distinguish between MARV, BDBV and SUDV-infected sera. The convalescent samples from all MARV, BDBV, and SUDV showed strong reactivity towards autologous antigens (FIG. 10). The MARV group showed significant increases against autologous GP-mucin (p<0.001), GPΔTM (insect, p<0.001), NP (p<0.001) and VP40 (p<0.001) when compared to negative controls. Significant reactivity was seen for BDBV sera samples against BDBV antigens GP-Mucin (p<0.05), GPΔTM (insect, p<0.05), NP (p>0.05) and VP40 (p<0.05). Similarly, the SUDV sera samples showed significant increases in antibody binding to SUDV GPΔTM (insect, p<0.05) and NP (p<0.005). However, the increase against SUDV VP40 was not statistically significant (Table 2). For the case of the rAd5-vaccinated individual, a robust antibody response was observed against EBOV GPΔTM (mammalian) but not against SUDV GP (Supplementary Figure). The recombinant rAd5 vaccine expressed EBOV and SUDV GP proteins, and most vaccinees produced antibodies against both proteins, as detected by ELISA (Ledgerwood, Costner et al. 2010).

We examined the cross-reactivity of the Ebola and Marburg convalescent sera against heterologous antigens (FIG. 11). Comparing all survivor sera, antibodies from the MARV infection cases were the least cross reactive with ebolavirus GPΔTM antigens, while only the BDBV-GP mucin interacted significantly (p<0.005) with MARV convalescent serum antibodies. The MARV sera did cross-react with other heterologous antigens, but to a much lower extent than BDBV and SUDV survivors. The SUDV sera exhibited significant levels of antibodies against RESTV GPΔTM (insect, p<0.05), RESTV NP (p<0.05), and TAFV NP (p<0.05). The BDBV presented the highest cross reactivity among filovirus antigens. Significant antibody binding was observed with EBOV NP (p<0.05), EBOV VP40 (p<0.05), SUDV NP (p<0.05), TAFV VP40 (p<0.05), RESTV GPΔTM (insect, p<0.05), and RESTV NP (p<0.05). Among the three antigens, the NP showed the highest levels of antibody cross-reactivity, followed by VP40, and GP proteins.

TABLE 3 Summary of recombinant proteins used in the microarray Species Proteins MARV GP-mucin, GPΔTM (insect), GPΔTM (mammalian), NP, VP40 EBOV GP-mucin, GPΔTM (insect), GPΔTM (mammalian), NP, VP40 BDBV GP-mucin, GPΔTM (insect), GPΔTM (mammalian), NP, VP40 SUDV GP-mucin, GPΔTM (insect), GPΔTM (mammalian), NP, VP40 TAFV GP-mucin, GPΔTM (mammalian), NP, VP40 RESTV GP-mucin, GPΔTM (insect), GPΔTM (mammalian), NP, VP40

Discussion

The data demonstrates that the compositions and methods provided herein can identify antibody responses by human survivors of Ebola and Marburg infection. Previous studies have reported that most antibody production in hosts was directed mainly towards GP, NP and VP40 proteins (Johnson, Wambui et al. 1986, Leroy, Baize et al. 2000, Lee, Fusco et al. 2008). Hence, for this example, these three proteins were used in a microarray application. The VP40 and NP were expressed as full length recombinant proteins. The GP proteins were produced in three different formats, namely GP-mucin, insect cell expressed GPΔTM, and mammalian cell expressed GPΔTM. The GP protein is extensively glycosylated (Lee, Fusco et al. 2008). We included both multi-domain GPΔTM, expressed as glycosylated proteins in insect or mammalian cell cultures, and single domain GP-mucins that were produced as non-glycosylated proteins in E. coli. The results presented here show that the mucin-like domain contributes substantially to human antibody, as polyclonal antibody recognition of the isolated GP mucin-like domain was comparable to the GPΔTM recombinant protein (see also, Kamata, Natesan et al. 2014).

Antibody responses to Ebola infections have been studied using ELISA (Nakayama, Yokoyama et al. 2010) (Prehaud, Hellebrand et al. 1998) (Saijo, Niikura et al. 2001) and Western blots (Leroy, Baize et al. 2000). Nakayama et aL has used recombinant GP from Ebola in ELISA to detect IgG and IgM from infected individuals. Neutralizing humoral responses to Ebola proteins by human survivors of SUDV (Gulu) infection by ELISA were also reported (Sobarzo, Perelman et al. 2012) (Sobarzo, Groseth et al. 2013), and viral cell cultures were used as antigens in an ELISA to study IgM and IgG responses (Macneil, Reed et al. 2011). Antibody cross-reactivity among Ebola and Marburg viruses is not well-characterized at the protein level. The sera samples were obtained from survivors of three different outbreaks that occurred in Uganda (Gulu-2000, Bundibugyo-2007 and Kabale-2012). The time of sera collection varied from one year (MARV), seven years (BDBV), and fourteen years (SUDV) after the outbreak of the disease. Two types of controls were used in this study. One group comprised of sera from healthy controls collected from the same area (Uganda) and the second group of sera collected from a completely different geographical region (USA). The background antibody levels in endemic areas were slightly elevated compared to sera obtained from non-endemic areas, emphasizing the need to include appropriate controls for correct interpretation of data.

The convalescent sera exhibited high levels of antibody binding for all antigens from the same species of filovirus that caused the infection (FIG. 9). Among the three antigens we tested, antibody levels were highest for NP, followed by GP and VP40. The NP protein is essential for replication of viral genome and formation of the nucleocapsid. Each virion contains about 3200 NP molecules (Bharat, Noda et al. 2012). The C-terminus region of the NP protein is highly antigenic and many epitopes have been identified in this region (Saijo, Niikura et al. 2001) (Changula, Yoshida et al. 2013). Hence, it is not surprising that NP elicits strong antibody responses in infected subjects, and our results confirm this observation and corroborate other reports (Sobarzo, Perelman et al. 2012). GP is the primary surface protein of the virion and several experimental vaccines and antibody-based therapeutics target GP. For the three different forms of recombinant GP (GP-mucin, GPΔTM-insect, GPΔTM-mammalian) used in our microarray, the level of antibody binding was similar (FIG. 13 or data not shown) for all six species except for insect cell produced BDBV GPΔTM, which showed higher binding than GP-mucin and GPΔTM-mammalian. Although the VP40 protein is the most abundant protein within the mature virus, our results show that the antibody response to VP40 in humans is lower than that obtained with NP and GP proteins. It is striking that 14 years after infection many individuals within the SUDV survivor group retain antibodies against filoviral proteins. Persistence of antifilovirus antibodies in long-term survivors of infection was previously reported (Wauquier, Becquart et al. 2009, Sobarzo, Ochayon et al. 2013).

Our study did not include human survivors from EBOV infection from the current West African outbreak due to the unavailability of samples. We did include in our study a serum sample from a human subject vaccinated with chimpanzee rAd5 Ebola vaccine developed by the National Institutes of Health (Ledgerwood, Costner et al. 2010). The vaccine was bivalent, expressing both EBOV and SUDV GP proteins. We found robust antibody response against EBOV GPΔTM (mammalian) but not against SUDV GP protein (FIG. 12). The absence and diminished response against GP proteins may be due to the presence of pre-existing neutralizing antibodies against the Ad5 vector. The capacity of pre-existing vector-specific humoral responses to interfere with efficacy of vaccines is well documented (Pine, Kublin et al. 2011, Ledgerwood, DeZure et al. 2014). In our previous study (Kamata, Natesan et al. 2014) of rhesus macques that were vaccinated with EBOV VLP and challenged with live EBOV, antibodies to GP presented the highest level of specificity in contrast to the human survivor serum antibodies, which showed appreciable amounts of cross-reactivity to heterologous GP proteins. Combined, these results suggest that for the GP antibody response, lower primate and humans may differ depending on how each were exposed to filoviruses. Nakayama et al. reported similar findings by comparing antibody responses to Ebola by mice and humans (Nakayama, Yokoyama et al. 2010).

Cross-reactivity towards heterologous antigens can be observed for all three groups of survivors (BDBV, SUDV and MARV). This is expected since there is considerable amount of protein sequence homology found between the five Ebola species (FIG. 8). The MARV species show the least homology to other members of Ebola. Our phylogenic analysis showed (FIG. 8) that NP is highly conserved among the filoviral species, hence may have common epitiopes that are present in all filovirus species. More than 30% of NP residues are conserved among all six species (FIG. 8). The conserved region of NP forms a condensed helix and may contain a structure that plays a role in virus replication (Bharat, Noda et al. 2012). A previous study (Changula, Yoshida et al. 2013) has identified epitopes in this region that cross react to all or several members of Filoviridae. However, the highly variable C terminal region of NP contains the highest number of antigenic regions. This example confirms that among the three antigens tested the NP showed the most cross-reactivity, in agreement with previous reports (Sobarzo, Groseth et al. 2013, Sobarzo, Ochayon et al. 2013, McElroy, Akondy et al. 2015).

This example provides the first report describing a multiplexed assay method and a multiplex protein microarray that was used to identify and analyze antibody specificity and cross-reactivity from human survivors of Ebola and Marburg. While the data suggests a considerable amount of variability in human antibody responses, it identifies GP as desirable targets for neutralizing antibodies (as GP are responsible for virus entry into cells). Nevertheless, the immune responses observed for VP40 and NP can also serve as suitable diagnostic indicators and biomarkers of infection. The disclosure thus provides for detection agents and methods that can effectively identify filoviral-specific antibodies in a sample, and further allows for the application of additional proteins or lysates from filovirus and other new viruses that may present outbreak concerns and issues of public health. Further, the methods and compositions disclosed herein can distinguish Ebola infections from diseases that mimic symptoms similar to that of filoviral infections, and can be used as a tool for seroepidemiological screening as well as for the diagnosis of filoviral infections in mammals.

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The invention claimed is:
 1. A detection agent comprising a substrate, one or more glycoproteins (GPs), one or more nucleoproteins (NP), and one or more virion protein 40 (VP40); the one or more GPs, the one or more NPs, and the one or more VP40s being attached to the substrate, immunoreactive as attached to the substrate, and from Marburg marburgvirus, Sudan ebolavirus, Zaire ebolavirus, Reston ebolavirus, Bundibugyo ebolavirus, or Taï Forest ebolavirus; at least one GP, at least one NP, and at least one VP40 being from different filoviruses; the one or more NPs comprising SEQ ID NO:4, SEQ ID NO:10, SEQ ID NO:16, SEQ ID NO:22, SEQ ID NO: 28, or SEQ ID NO:34; the one or more VP40s comprising SEQ ID NO:2, SEQ ID NO:8, SEQ ID NO:14, SEQ ID NO:20, SEQ ID NO:26, or SEQ ID NO:32; and the one or more GPs comprising a GP-mucin domain or a GP ectodomain (GPΔTM) and the one or more GPs further comprising SEQ ID NO:6, SEQ ID NO:12, SEQ ID NO:18, SEQ ID NO:24, SEQ ID NO:30, or SEQ ID NO:36.
 2. The detection agent of claim 1 being selected from the group consisting of a microarray, microparticles, and nanoparticles.
 3. The detection agent of claim 1 being a microarray.
 4. A method for detecting the presence of a filovirus-specific antibody in a biological sample obtained from a subject comprising: (a) incubating the biological sample with the detection agent of claim 1 under conditions that allow binding of the filovirus-specific antibody to the detection agent; and (b) detecting the filovirus-specific antibody bound to detection agent.
 5. The method of claim 4, the detection agent comprising the fragment of the GP-mucin.
 6. The method of claim 4, the detection agent comprising the GPΔTM or fragment thereof.
 7. A method for detecting filovirus antibodies in a subject, comprising: a) incubating a sample obtained from the subject with the detection agent of claim 1 under conditions that facilitate antigen-antibody binding; and b) detecting the bound antibody in a).
 8. The method of claim 7, the detection agent comprising the fragment of the GP-mucin.
 9. The method of claim 7, the detection agent comprising the GPΔTM or fragment thereof.
 10. A kit comprising: the detection agent of claim 1; at least one reagent that can detect a filovirus-specific antibody bound to the detection agent of claim 1; and instructions for use of the kit.
 11. The detection agent of claim 1 comprising the fragment of the GP-mucin.
 12. The detection agent of claim 1 comprising the GPΔTM or fragment thereof.
 13. The detection agent of claim 1, comprising a non-glycosylated GP-mucin.
 14. The method of claim 4, the detection agent comprising a non-glycosylated GP-mucin.
 15. The method of claim 7, the detection agent comprising a non-glycosylated GP-mucin.
 16. The kit of claim 10, the detection agent comprising a non-glycosylated GP-mucin.
 17. A detection agent comprising a substrate, a first nucleoprotein (NP) comprising SEQ ID NO:4, a second NP comprising SEQ ID NO:10, a third NP comprising SEQ ID NO:16, a fourth NP comprising SEQ ID NO:22, a fifth NP comprising SEQ ID NO: 28, a sixth NP comprising SEQ ID NO:34, a first virion protein 40 (VP40) comprising SEQ ID NO:2, a second VP40 comprising SEQ ID NO:8, a third VP40 comprising SEQ ID NO:14, a fourth VP40 comprising SEQ ID NO:20, a fifth VP40 comprising SEQ ID NO:26, a sixth VP40 comprising SEQ ID NO:32, a first glycoprotein (GP) comprising SEQ ID NO:6, a second GP comprising SEQ ID NO:12, a third GP comprising SEQ ID NO:18, a fourth GP comprising SEQ ID NO:24, a fifth GP comprising SEQ ID NO:30, and a sixth GP comprising SEQ ID NO:36; the first through sixth NPs, the first through sixth VP40s, the first through sixth GPs being attached to the substrate and immunoreactive as attached to the substrate; the first through sixth GPs comprising a GP-mucin domain or a GP ectodomain (GPΔTM).
 18. A method for detecting the presence of a filovirus-specific antibody in biological sample obtained from a subject comprising: (a) incubating the biological sample with the detection agent of claim 17 under conditions that allow binding of the filovirus-specific antibody to the detection agent; and (b) detecting the filovirus-specific antibody bound to detection agent.
 19. A method for detecting filovirus antibodies in a subject, comprising: a) incubating a sample obtained from the subject with the detection agent of claim 17 under conditions that facilitate antigen-antibody binding; and c) detecting the bound antibody in b). 